Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia, and meningitis in the USA despite chemoprophylaxis strategies for preventing this infection recommended by the CDC. Although serotype III GBS account for the majority of infections, newer serotypes are emerging, eg. type V. We have demonstrated the importance of the capsular polysaccharide (CPS) to GBS virulence. Using transposon mutagenesis we identified a 20Kb region of the type III chromosome responsible for the biosynthesis of the type III capsular polysaccharide. 16 genes have now been identified by sequencing analysis which are organized according to function, putatively involved in regulation, chain length determination, export, pentasaccharide repeating unit synthesis, and sialic acid synthesis. The genetic organization is similar to that observed for the genes encoding complex polysaccharide synthesis in other gram (+) and gram (-) bacteria. Several cps genes share homology with cps genes in other organisms, although we have identified unique genes as well, which require further characterization. We have identified the first sialic acid (SA) synthesis genes in a gram (+) bacteria which share functional homology with the neu genes responsible for sialic acid and CPS synthesis in E. coli K1. This proposal seeks to extend our investigation of the type III cps gene loci. The type III cps genes are transcribed as a large polycistronic message and therefore to investigate their function we will need to develop non-polar mutations in each of the 16 genes. These mutants will be critical for investigating gene function within GBS and will be derived in aim 1, using contemporary genetic methods we developed for this organism. Each wild type gene will be cloned during the mutagenesis process, which will be important for subsequent complementation studies. Aim 2 will investigate the unique SA synthesis genes, exploiting our recent observations that they complement homologue mutations in E.coli K1. During this investigation we will perform complementation analysis of the non-polar mutants with the SA synthesis genes from E.coli, will confirm the biochemical function of each gene, and identify the sialyltransferase gene responsible for linking sialic acid to CPS. Aims 3 and 4 will begin to characterize mutants in the putative glycosyltransferases, chain length, and export loci using complementation with homologue genes from S. pneumoniae type 14 and biochemical analysis, to identify, their functional role in cps synthesis. These studies should enhance our understanding of the genetic basis of CPS expression in GBS and other encapsulated gram (+) pathogenic bacteria. They may also provide clues to the evolution of new GBS serotypes which are now causing human infections.